Host Cell Oxidative Stress Promotes Intracellular Fluoroquinolone Persisters of Streptococcus pneumoniae

ABSTRACT Bacterial persisters represent a small subpopulation that tolerates high antibiotic concentrations without acquiring heritable resistance, and it may be generated by environmental factors. Here, we report the first antibiotic persistence mechanism in Streptococcus pneumoniae, which is induced by oxidative stress conditions and allows the pneumococcus to survive in the presence of fluoroquinolones. We demonstrated that fluoroquinolone persistence is prompted by both the impact of growth arrest and the oxidative stress response induced by H2O2 in bacterial cells. This process protected pneumococci against the deleterious effects of high ROS levels induced by fluoroquinolones. Importantly, S. pneumoniae develops persistence during infection, and is dependent on the oxidative stress status of the host cells, indicating that its transient intracellular life contributes to this mechanism. Furthermore, our findings suggest persistence may influence the outcome of antibiotic therapy and be part of a multistep mechanism in the evolution of fluoroquinolone resistance. IMPORTANCE In S. pneumoniae, different mechanisms that counteract antibiotic effects have been described, such as vancomycin tolerance, heteroresistance to penicillin and fluoroquinolone resistance, which critically affect the therapeutic efficacy. Antibiotic persistence is a type of antibiotic tolerance that allows a bacterial subpopulation to survive lethal antimicrobial concentrations. In this work, we used a host-cell infection model to reveal fluoroquinolone persistence in S. pneumoniae. This mechanism is induced by oxidative stress that the pneumococcus must overcome to survive in host cells. Many fluoroquinolones, such as levofloxacin and moxifloxacin, have a broad spectrum of activity against bacterial pathogens of community-acquired pneumonia, and they are used to treat pneumococcal diseases. However, the emergence of fluoroquinolone-resistant strains complicates antibiotic treatment of invasive infections. Consequently, antibiotic persistence in S. pneumoniae is clinically relevant due to prolonged exposure to fluoroquinolones likely favors the acquisition of mutations that generate antibiotic resistance in persisters. In addition, this work contributes to the knowledge of antibiotic persistence mechanisms in bacteria.

shown. Percentage values were obtained from data shown in panels A, C, E, G, I, K and M and represent the mean ± SEM of at least three replicates. Statistically significant differences were determined using the two-tailed test and are indicated as p < 0.05 (*), p < 0.01 (**) or p < 0.0001 (****).

FIG S2
Susceptibility to fluoroquinolones is restored in persisters after regrowth. To confirm that FQ persistence is a transient phenotype, the R801 strain was grown in BHI to mid-log phase and exposed to 20 mM H2O2 for 30 minutes before adding either 6 µg/ml levofloxacin (A), 5 µg/ml moxifloxacin (B), or 2.5 µg/ml ciprofloxacin (C) for 5 h. After 5 h of antibiotic treatment, samples were removed at the indicated time points. Bacterial cells were collected by centrifugation, washed in PBS, resuspended, and seeded into blood agar plates to determine bacterial survival. Aliquots of each culture were regrown in BHI to mid-log phase and exposed again with the antibiotic used initially for 5 h, and bacterial cells were treated as mentioned to determine CFU. The percentage of antibiotic persisters in H2O2-treated or non-treated cells is shown and represents the mean ± SEM of at least three replicates. Statistically significant differences were determined using the two-tailed test and are indicated as p < 0.05 (*), p < 0.01 (**) or p < 0.0001 (****).

FIG S3
FQ persistence under different stress conditions. (A) FQ persistence is poorly induced by acidic stress conditions. The wt strain was grown in BHI to mid-log phase before being exposed to either 20 mM H2O2 for 30 minutes or MD medium (pH 5.2) for 2h. Bacterial cells were collected by centrifugation, washed with PBS, resuspended in BHI and exposed to 6 µg/ml levofloxacin for 5 h. Non-treated cells were used as control. The number of viable cells was measured as described in Fig S1. Percentages were calculated with the CFU/ml values shown in the b and c panels. (B) CFU/ml values correspond to cultures without antibiotic treatment. (C) CFU/ml values corresponding to levofloxacin-treated cultures after 5 h of exposure. (D) FQ persistence mechanism is dependent on protein synthesis. The wt strain was grown in BHI to mid-log phase and exposed to either 2 µg/ml chloramphenicol (a typical protein-synthesis inhibitor) for 1 h, 20 mM H2O2 for 30 minutes, or 2 µg/ml chloramphenicol for 1 h followed by the addition of 20 mM H2O2 for 30 minutes. Posteriorly, bacterial cells were collected by centrifugation, washed with PBS, resuspended in BHI and exposed to 6 µg/ml levofloxacin for 5 h. Then, the number of viable cells was measured as described in Fig S1. The percentage of levo-persisters in cultures treated with different stressors was calculated with the CFU/ml values shown below. (E) CFU/ml values were obtained at different time points in cultures after stressor exposure but without levofloxacin treatment. (F) CFU/ml values were obtained at different time points in cultures following stressor exposure and treatment with 6 µg/ml levofloxacin for 5 h. For all panels, values represent the mean ± SEM of at least three replicates. Statistically significant differences were determined using the twotailed test and are indicated as p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****).

FIG S4 NAC treatment inhibits H2O2 production in S. pneumoniae. (A)
The wt strain was grown in BHI until the mid-log phase and exposed to either 20 mM H2O2 for 30 minutes, 10 mM NAC (inhibitor of ROS production) for 1 h, or 10 mM NAC for 1 h followed by the addition of 20 mM H2O2 for 30 minutes. The H2O2 concentration was measured by the horseradish peroxidase method. Values are expressed in µM and normalized against 1x10 6 viable cells. (B) The bacterial cells were cultured as mentioned in panel a. The number of viable cells was measured as described in Fig  S1. (C) The bacterial cells were cultured as mentioned in panel a. Posteriorly, each culture was exposed to 6 µg/ml levofloxacin for 5h. The number of viable cells was measured as described in Fig S1. The data shown in the a-b panels correspond to the determination of the percentage of levo-persisters shown in Fig 1. Values represent the mean ± SEM of at least three replicates. Statistically significant differences were determined using the two-tailed test and are indicated as p < 0.05 (*), p < 0.01 (**) or p < 0.0001 (****).

FIG S5 H2O2 treatment increases the doubling time of the pneumococcal cells. (A)
The wt strain was grown in BHI to mid-log phase and then exposed to either 20 mM H2O2, or 2 µg/ml chloramphenicol, or 2 µg/ml chloramphenicol for 1 h followed by the addition of 20 mM H2O2. In parallel, bacterial cells were collected by centrifugation and resuspended in MD at pH 5.2. All cultures were diluted 1/10 in the same bacterial media, and optical density at 600 nm was measured at different time points to draw the corresponding growth curves. (B) The doubling time of the wt strain cultured under different stress conditions was determined using the data shown in panel a, and it was calculated using the number of CFU/ml obtained at 0 and 5 h of exposure. Values represent the mean ± SEM of at least three replicates. Statistically significant differences were determined using the two-tailed test and are indicated as p < 0.0001 (****).

FIG S6
The ∆spxB, ∆sodA, and ∆tpxD mutants are more susceptible to H2O2 and generate fewer FQ persisters than the wt strain. (A) The ∆spxB, ∆sodA, ∆tpxD and wt strains were grown in BHI to mid-log phase and exposed to 20 mM H2O2 for 30 minutes. The number of viable cells was measured as described in Fig S1. (B) The percentage of bacterial survival was calculated with the data shown in panel A. To determine FQ persistence, the ∆spxB, ∆sodA, ∆tpxD and wt strains were grown in BHI to mid-log phase without any treatment (C) or exposed to 20 mM H2O2 for 30 minutes (D). Bacterial cells were collected by centrifugation, resuspended in BHI and exposed to 6 µg/ml levofloxacin for 5 h. The number of viable cells was measured as described in Fig S1. These CFU/ml values were used to determine the percentage of levopersisters shown in Fig 3. Values represent the mean ± SEM of at least three replicates. Statistically significant differences were determined using the two-tailed test and are indicated as p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****).  the wt strain at an MOI of 30:1 (bacterial: host cells). At different time points, samples were removed and treated with 10 µM H2DCFDA and 50 µg/ml propidium iodide for 30 minutes. Then, cells were analysed by flow cytometry to quantify ROS levels. The fold changes of ROS levels were expressed as fluorescence intensity of DCF(+)/IP(-)-cells infected at indicated times. To determine FQ persistence in host cells, the A549 pneumocytes (D) and Raw 264.7 macrophages (E) were pre-treated with either 5 mM or 10 mM NAC, respectively, and infected with the wt strain using an MOI of 30:1 (bacteria: host cells). Non-NAC-treated cells were used as control. The differentiated PLB-985 and PLB-985-KO (nox2 mutant with decreased ROS production) neutrophils (F) were infected with the wt strain using an MOI of 30:1 (bacteria: host cells). Bacterial survival progression was monitored using a typical protection assay, as described in Fig S7, in which gentamicin was used as an extracellular antibiotic to kill nonendocytosed/non-phagocyted pneumococci. After 6 µg/ml levofloxacin treatment, samples were taken at different times according to the endocytic/phagocytic capacity of host cells. Cells were lysed by centrifugation, and CFU was determined by incubation of these samples on blood agar plates at 37˚C for 16 h. The CFU values were used to calculate the FQ persistence percentage shown in Fig 4. Values represent the mean ± SEM of at least three replicates. Statistically significant differences were determined using the two-tailed test and are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****).

FIG S9
The PBL-985 and PLB-985-KO cells were efficiently differentiated after DMSO treatment. The PLB-985 and PLB-985-KO cells were cultured with RPMI culture medium supplemented with 5% FBS, 1% penicillin/streptomycin, and 1.3% DMSO. Cells were differentiated into neutrophils after 5 days. As a control, undifferentiated PLB-985 and PLB-985-KO cells were cultured in the absence of DMSO. To quantify the cell differentiation and cell death percentages, cells were stained with anti-CD11b (A) and propidium iodide (B) and analysed by flow cytometry during the process on days 0, 3 and 5. Values represent the mean ± SEM of at least three replicates. Statistically significant differences were determined using the two-tailed test and are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****).

FIG S10
The spxB, sodA, and tpxD genes are involved in the induction of FQ persistence in host cells. The A549 (A and B), Raw 264.7 (C and D) and PLB-985 (E and F) cells were infected with the ∆spxB, ∆sodA, ∆tpxD and wt strains using an MOI of 30:1 (bacteria: host cells). The number of levo-persisters was determined as described in the Fig 4 legend. As a control, host cells were not treated with levofloxacin. The CFU values obtained in all panels were used to calculate the percentage of FQ persisters shown in Fig 4. Values represent the mean ± SEM of at least three replicates. Statistical significance was determined using the two-tailed test and indicated as p < 0.05 (*), p < 0.01 (**), or p < 0.0001 (****).